Effects of the E-liquid Flavoring Agents Vanillin and Ethyl Vanillin in Human Proximal Tubule Epithelial Cells

Abstract

<b>Abstract ID 21988</b> <b>Poster Board 425</b> E-cigarette usage and vaping have become increasingly popular over recent years, especially among teenagers and young adults. There are thousands of e-liquid flavors on the market, but the effects of inhaling e-liquid flavorings have not been well-characterized systemically. The lung remains the major focus of research, but some research shows that organs such as the kidney may be affected by e-liquid flavoring inhalation. The aldehydes vanillin (VAN) and ethyl vanillin (ETH VAN) are two popular flavoring ingredients found in e-liquids. The aim of this study is to evaluate VAN and ETH VAN cytotoxicity in renal proximal tubular epithelial cells. All studies were conducted using human renal proximal tubular cells (HK-2). Cells were obtained from ATCC and cultured according to vendor specifications. HK-2 cells were plated, equilibrated for 48 h, and treated with 0 (200 proof ethanol) or 100-1000 uM VAN or ETH VAN for 24 or 48 h. Viability was quantitated using the MTT assay and conversion to formazan. Cell Countess was performed to evaluate trypan blue exclusion and cell membrane leakage. Western blot analysis probed for oxidative stress as 4-hydroxynonenal (4-HNE) adducted proteins. Cell stress was evaluated as protein expression of: LC3B-I, LC3B-II, and caspase-3. Mitochondrial function was evaluated using an Agilent XFp Seahorse analyzer. Results were obtained from at least 4 independent experiments using different cell passages. Statistical differences between groups were analyzed using One-Way ANOVA followed by post hoc Tukey test at a 95% confidence interval. VAN and ETH VAN were not cytotoxic relative to control based on MTT assay after 24 h exposure, and ETH VAN showed an elevated conversion to formazan at 24 h. At 48 h, VAN treatment showed significantly decreased (p&lt;0.05) conversion to formazan at 1000 uM concentration when compared to control. ETH VAN showed significantly decreased (p&lt;0.05) conversion to formazan at 1000 uM concentration when compared to control and 100-500 uM treatment groups at 48 h. Countess trypan blue exclusion assay showed no significant difference between groups at all concentrations at 24 or 48 h. No significant 4-HNE adduction of proteins was observed for either VAN or ETH VAN at any concentration when compared to control. Autophagy was evaluated as the ratio of LC3B-II to LC3B-I. The ratio of LC3B-II to LC3B-I was not significantly different for either VAN or ETH VAN, but an increasing trend in ratio was observed. Apoptosis probing showed no significant increase in cleaved caspase-3 for either VAN or ETH VAN, however an increasing trend was observed for ETH VAN. Seahorse analyzer assay of mitochondrial oxygen consumption rate (OCR) showed that basal respiration significantly increased (p&lt;0.05) at 1000 uM VAN 24 h when compared to control and the 100 uM concentration. ATP production was significantly increased (p&lt;0.05) between 100 uM and 1000 uM concentrations for VAN 24 h. Basal respiration, non-mitochondrial respiration, maximal respiration, and proton (H+) leak all significantly increased (p&lt;0.05) at 1000 uM ETH VAN 24 h when compared to control and the 100 uM concentration. Spare respiratory capacity significantly increased (p&lt;0.05) between the control and 1000 uM ETH VAN 24 h group. These findings indicate that VAN and ETH VAN exposure induce changes within the mitochondria based on Seahorse assay and 48 h MTT assay. Further studies are needed in order to characterize specific changes within the mitochondria for these flavoring agents. Supported by NIH Grant P20GM103424; AC supported by WV NASA Graduate Research Fellowship.